This invention relates to nucleic acid and amino acid sequences of a novel human eosinophil-derived basic protein and to the use of these sequences in the diagnosis, prevention, and treatment of disease.
Stem cells are progenitor blood cells which differentiate to mature white blood cells, rid blood cells, and platelets. Stem cells are found in adult bone marrow, in fetal liver and spleen, and in blood collected from the umbilical cord after the birth of a baby.
Eosinophil growth and differentiation from stem cells is regulated by hematopoietic growth factors including granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5). IL-5 is a potent eosinophil differentiation and activation factor, while GM-CSF and IL-3 also increase the production of other myeloid cells.
Eosinophils are white blood cells which are sub-classified as granulocytes due to the presence of large, coarse membrane-bound cytoplasmic granules. These granules contain proteins and other compounds which carry out a variety of inflammatory and immune functions. In response to chemotactic factors, eosinophils migrate through blood vessel walls and through tissues to the site where they are needed. There the contents of the granules are released in response to specific stimuli. Eosinophil granule release is stimulated by immunoglobulin E (IgE)-mediated hypersensitivity reactions such as parasitic infections and type I allergic reactions. Such type I allergic reactions include asthma and allergic rhinitis.
A variety of eosinophil-derived basic proteins (EDBPs) are released from eosinophil granules. These cytotoxic proteins disrupt membrane surfaces and lyse cells. EDBPs are thus potent anti-parasitic and anti-bacterial agents, however, EDBPs may also damage host tissues. For instance, the cardiovascular damage associated with chronic hypereosinophilia has been attributed in part to secreted EDBPs. EDBPs have been shown to damage respiratory epithelial cells, and have been implicated in the increase in bronchial hyperreactivity frequently observed in asthma patients. A significant correlation exists between the intensity of bronchial hyperreactivity and the levels of EDBPs in blood and bronchoalveolar lavage (BAL) fluid from asthmatics.
Eosinophil granule major basic protein (MBP), one of the most extensively characterized EDBPs, is a potent toxin against helminths, protozoa, bacteria and other cells. MBP also causes epithelial desquamization and ciliostasis, effects that mimic the pathology of asthma (Gleich, G. J. et al. (1988) J. Allergy Clin. Immun. 81:776-781). MBP is found in sputa and on damaged bronchial tissues of asthma patients (Frigas, E. et al. (1981) Mayo Clin. Proc. 56:345; Filley, W. V. et al. (1982) Lancet 2:11).
Along with its cytolytic properties, MBP also possesses noncytolytic proinflammatory properties, many of which are associated with late phase reactions of allergic disease. The release of histamine and leukotriene C4 from basophils is stimulated by MBP and further enhanced by the cytokines IL-3, IL-5 and GM-CSF (Sarmiento, E. U. et al. (1995) J. Immunol. 155:2211-2221). MBP also stimulates neutrophil activation and degranulation, including the release of superoxide anion (O2-) and lysozyme (Moy, J. N. et al. (1990) J. Immunol. 145:2626-2632), and platelet activation (Rohrbach, M. S. et al. (1990) J. Exp. Med. 172:1271-1274). MBP also induces further eosinophil degranulation (Kita, H. et al. (1995) J. Immunol. 154:4749-4758).
The cDNA for MBP encodes a 25 kdal preproprotein molecule of 222 amino acids, which includes a predicted 15 amino acid leader peptide, a 90 amino acid acidic pro domain and a 117 amino acid mature polypeptide (Popken-Harris, P. et al. (1995) J. Immunol. 155:1472-1480). The pro domain of MBP contains a heterogeneous population of O- and N-linked glycosyl modifications and has an isoelectric point (pI) of approximately 4.9. The 14 kdal mature MBP contains two disulfide bridges and five free cysteine residues (Oxvig, C. et al. (1994) FEBS Lett. 341:213-217).
The negatively-charged pro domain appears to interact with the positively-charged mature MBP. This interaction is proposed to inhibit the activity of mature MBP thus protecting the developing eosinophil from damage by MBP during granule processing (Popken-Harris, et al. (1995), supra). Mature MBP, but not proMBP, reacts readily with acidic lipids and disorders lipid bilayers resulting in the lysis of liposomes (Abu-Ghazaleh, R. I. et al. (1992) J. Membr. Biol. 128:153-164). Unlike mature MBP, proMBP does not stimulate basophil histamine release or neutrophil superoxide generation; in fact, proMBP is an inhibitor of these MPB-stimulated activities (Popken-Harris, et al. (1995), supra).
ProMBP is expressed in placental X cells and is found in the sera of pregnant women (Wagner, J. M. et al. (1994) Placenta 15:625-640). Levels of proMBP peak before labor and rapidly decline after delivery (Maddox, D. E. et al. (1983) J. Exp. Med. 158:1211-1216). ProMBP of placental origin is heavily glycosylated and circulates in disulfide-bridged complexes with pregnancy-associated plasma protein A (PAPP-A), angiotensinogen, and complement C3dg (Oxvig C. et al. (1993) J. Biol. Chem. 268:12243-12246; Oxvig C. et al (1995) J. Biol. Chem. 270:13645-13651). Low serum levels of PAPP-A in the first trimester have been linked to fetal chromosomal abnormalities (Brambati, M. C. (1993) Br. J. Obstet. Gynaecol. 100:324-326). A high molecular weight (HMW) form of angiotensinogen has been found in moderate quantities in plasma from pregnant women and in high quantities in hypertensive pregnant women (Tewksbury, D. A. et al. (1989) Am. J. Hypertens. 2:411-413). Oxvig and colleagues (1995, supra) suggest that this HMW angiotensinogen is actually the proMBP:angiotensinogen complex.
The discovery of polynucleotides encoding a novel EDBP-like molecule, and the molecule themselves, satisfies a need in the art by providing a new means for the diagnosis, prevention, or treatment of diseases and conditions associated with eosinophil accumulation and granule release including late-phase allergic/inflammatory reactions, eosinophilias, parasitic infections, and conditions associated with placental derived-EDBP accumulation in pregnancy.
The present invention features a novel basic protein derived from IL-5 cultured umbilical cord blood cells, hereinafter designated as EBPH and characterized as having chemical and structural homology to eosinophil granule MBPs from human and guinea pig.
Accordingly, the invention features a substantially purified human EBPH having the structural characteristics of the MBPs above and as shown in amino acid sequence, SEQ ID NO:1.
One aspect of the invention features isolated and substantially purified polynucleotides that encode EBPH. In a particular aspect, the polynucleotide is the nucleotide sequence of SEQ ID NO:2.
The invention also features a polynucleotide sequence comprising the complement of SEQ ID NO:2 or variants thereof. In addition, the invention features polynucleotide sequences which hybridize under stringent conditions to SEQ ID NO:2.
The invention additionally features nucleic acid sequences encoding polypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments, portions or antisense molecules thereof, and expression vectors and host cells comprising polynucleotides that encode EBPH. The present invention also features antibodies which bind specifically to EBPH, and pharmaceutical compositions comprising substantially purified EBPH. The invention also features agonists and antagonists of EBPH.